Pfizer Global Research & Development, Andover, MA 01810, USA.
Anal Biochem. 2010 Nov 15;406(2):113-23. doi: 10.1016/j.ab.2010.06.044. Epub 2010 Jul 23.
The degradation of articular cartilage by aggrecanases (ADAMTS-4 and ADAMTS-5) plays a significant role in the pathology of osteoarthritis (OA). To monitor aggrecanase activity in OA, we have developed a sensitive, accurate, and versatile assay for detection of two specific cleavage sites on aggrecan. The assay uses an immunoaffinity-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect cleavage at the (374)ARGS site and the (1820)AGEG site. The dynamic range of the assay is more than three orders of magnitude, with interassay precision less than 15%. It has been successfully applied to various biological fluids and species, including rat, bovine, dog, and human. The assay has been analytically qualified for use in human urine and synovial fluid (SF). The limits of detection (LODs) for ARGS in urine and SF are 2.5 and 10 pg/ml, respectively, whereas the LOD for AGEG is 20 pg/ml in SF. Analysis of these biomarkers from OA subjects and normal healthy volunteers revealed a significant elevation of both markers in OA. Similarly, in a rat model of cartilage degradation, both ARGS and AGEG were elevated, demonstrating the utility of these biomarkers for translational research. These data suggest that the ARGS and AGEG biomarkers developed have potential as measures of aggrecanase activity in OA and may contribute to our understanding of OA pathology.
聚集蛋白聚糖酶（ADAMTS-4 和 ADAMTS-5）对关节软骨的降解在骨关节炎（OA）的病理学中起着重要作用。为了监测 OA 中的聚集蛋白聚糖酶活性，我们开发了一种灵敏、准确且多功能的检测方法，用于检测聚集蛋白聚糖上两个特定的切割位点。该测定法使用基于免疫亲和的液相色谱-串联质谱（LC-MS/MS）方法来检测（374）ARGS 位点和（1820）AGEG 位点的切割。该测定法的动态范围超过三个数量级，批间精密度小于 15%。它已成功应用于各种生物流体和物种，包括大鼠、牛、狗和人。该测定法已在人尿液和滑液（SF）中进行了分析验证。ARG 在尿液和 SF 中的检测限（LOD）分别为 2.5 和 10 pg/ml，而 AGEG 在 SF 中的 LOD 为 20 pg/ml。对 OA 患者和正常健康志愿者的这些生物标志物的分析显示，OA 患者的两种标志物均显著升高。同样，在软骨降解的大鼠模型中，ARG 和 AGEG 均升高，证明了这些生物标志物在转化研究中的应用。这些数据表明，开发的 ARGS 和 AGEG 生物标志物具有作为 OA 中聚集蛋白聚糖酶活性的测量指标的潜力，并可能有助于我们对 OA 病理学的理解。
